Nightshift work and genome-wide DNA methylation

<div><p>The negative health effects of shift work, including carcinogenesis, may be mediated by changes in DNA methylation, particularly in the circadian genes. Using the Infinium HumanMethylation450 Bead Array (Illumina, San Diego, CA), we compared genome-wide methylation between 65 actively working dayshift workers and 59 actively working nightshift workers in the healthcare industry. A total of 473 800 loci, including 391 loci across the 12 core circadian genes, were analyzed to identify methylation markers associated with shift work status using linear regression models adjusted for gender, age, body mass index, race, smoking status and leukocyte cell profile as measured by flow cytometry. Analyses at the level of gene, CpG island and gene region were also conducted. To account for multiple comparisons, we controlled the false discovery rate (FDR ≤0.05). Significant differences between nightshift and dayshift workers were found at 16 135 of 473 800 loci, across 3769 of 20 164 genes, across 7173 of 22 721 CpG islands and across 5508 of 51 843 gene regions. For each significant loci, gene, CpG island or gene region, average methylation was consistently found to be decreased among nightshift workers compared to dayshift workers. Twenty-one loci located in the circadian genes were also found to be significantly hypomethylated among nightshift workers. The largest differences were observed for three loci located in the gene body of <i>PER3</i>. A total of nine significant loci were found in the <i>CSNK1E</i> gene, most of which were located in a CpG island and near the transcription start site of the gene. Methylation changes in these circadian genes may lead to altered expression of these genes which has been associated with cancer in previous studies. Gene ontology enrichment analysis revealed that among the significantly hypomethylated genes, processes related to host defense and immunity were represented. Our results indicate that the health effects of shift work may be mediated by hypomethylation of a wide variety of genes, including those related to circadian rhythms. While these findings need to be followed-up among a considerably expanded group of shift workers, the data generated by this study supports the need for future targeted research into the potential impacts of shift work on specific carcinogenic mechanisms.</p></div

Characteristics of participants randomized in the crossover trial of glucosamine and chondroitin at baseline (n = 18).

<p>¹Mean (SD)</p><p>²Measured by Dual X-ray Absorptiometry (DEXA)</p><p>³Geometric mean (geometric SD)</p><p>Characteristics of participants randomized in the crossover trial of glucosamine and chondroitin at baseline (n = 18).</p

Top 100 (of 508) individual protein antibodies significantly different after glucosamine and chondroitin supplementation versus placebo intervention periods (n = 18).

<p>¹Indicates that protein was in the GO (Gene Ontology) or KEGG (Kyoto Encyclopedia of Genes and Genomes) Cytokines pathway</p><p>²Information pertaining to function is derived from PubMed Gene and/or UniProtKB unless otherwise noted</p><p>³Values >1 indicate greater antibody expression after supplementation with G&C compared to placebo; values <1 indicate lower expression</p><p>*All proteins listed were statistically significant with Bonferroni correction (<i>P</i> <1.70x10^-5)</p><p>Top 100 (of 508) individual protein antibodies significantly different after glucosamine and chondroitin supplementation versus placebo intervention periods (n = 18).</p

Significantly different pathways comparing glucosamine and chondroitin supplementation to placebo via gene-set enrichment analysis in Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases (n = 18).

<p>¹ Among the total number of genes in the pathway, number of unique genes in our array data</p><p>² Total number of corresponding antibody probes in the assay</p><p>³ Among the significant probes, via paired t-test comparing G&C to placebo, the number that have an effect size >0</p><p>⁴ Among the significant probes, via paired t-test comparing G&C to placebo, the number that have an effect size <0</p><p>⁵ GO = Gene Ontology; KEGG = Kyoto Encyclopedia of Genes and Genomes</p><p>*All pathways listed were statistically significant with Bonferroni correction (<i>P</i> <3.85x10^-4 for KEGG pathway; <i>P</i><5.18x10^-5 for GO pathway)</p><p>Significantly different pathways comparing glucosamine and chondroitin supplementation to placebo via gene-set enrichment analysis in Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases (n = 18).</p

Fatty acids in non-alcoholic steatohepatitis: Focus on pentadecanoic acid

<div><p>Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease and ranges from isolated steatosis to NASH. To determine whether circulating fatty acids could serve as diagnostic markers of NAFLD severity and whether specific fatty acids could contribute to the pathogenesis of NASH, we analyzed two independent NAFLD patient cohorts and used the methionine- and choline-deficient diet (MCD) NASH mouse model. We identified six fatty acids that could serve as non-invasive markers of NASH in patients with NAFLD. Serum levels of 15:0, 17:0 and 16:1n7t negatively correlated with NAFLD activity scores and hepatocyte ballooning scores, while 18:1n7c serum levels strongly correlated with fibrosis stage and liver inflammation. Serum levels of 15:0 and 17:0 also negatively correlated with fasting glucose and AST, while 16:1n7c and 18:1n7c levels positively correlated with AST and ferritin, respectively. Inclusion of demographic and clinical parameters improved the performance of the fatty acid panels in detecting NASH in NAFLD patients. The panel [15:0, 16:1n7t, 18:1n7c, 22:5n3, age, ferritin and APRI] predicted intermediate or advanced fibrosis in NAFLD patients, with 82% sensitivity at 90% specificity [AUROC = 0.92]. 15:0 and 18:1n7c were further selected for functional studies <i>in vivo</i>. Mice treated with 15:0-supplemented MCD diet showed reduced AST levels and hepatic infiltration of ceroid-laden macrophages compared to MCD-treated mice, suggesting that 15:0 deficiency contributes to liver injury in NASH. In contrast, 18:1n7c-supplemented MCD diet didn’t affect liver pathology. In conclusion, 15:0 may serve as a promising biomarker or therapeutic target in NASH, opening avenues for the integration of diagnosis and treatment.</p></div

Fatty acids that showed significantly different levels in serum between patients with NAFLD activity scores (NAS) 1–4 (n = 68) and patients with NAS ≥5 (n = 38).

<p>(A) Saturated fatty acids (SFAs), pentadecanoic (15:0) and heptadecanoic (17:0) acid, (B) monounsaturated fatty acids (MUFAs), palmitelaidic (16:1n7t), palmitoleic (16:1n7c) and vaccenic (18:1n7c) acid. Fatty acids were measured as percentage of the phospholipid fraction. P-value was calculated using two-tailed student’s T-test.</p

Characteristics of the patient population.

<p>Characteristics of the patient population.</p

Pentadecanoic acid (15:0), vaccenic acid (18:1n7c) and docosapentaenoic acid (22:5n3) serum levels in patients.

<p>Isolated steatosis (n = 27), NASH patients with fibrosis stage 0–1 (n = 45) and NASH patients with fibrosis stage 2–4 (n = 34). Fatty acids were measured as percentage of the phospholipid fraction. Statistical significance was done using ANOVA test and then pairwise comparison using Tukey’s test was performed.</p

Receiver operating characteristic (ROC) analysis.

<p>(A) based on NAFLD activity scores (NAS)–patients with NAS 1–4 versus patients with NAS ≥5. (B) Based on fibrosis scores—patients with fibrosis 0–1 versus patients with fibrosis 2–4.</p

Correlation of fatty acids with NASH parameters.

<p>(A) Correlation of pentadecanoic (15:0), heptadecanoic (17:0) acid and palmitelaidic (16:1n7t) with NAFLD activity scores (NAS), (B) correlation of pentadecanoic (15:0), heptadecanoic (17:0) acid and palmitelaidic (16:1n7t) with hepatocyte ballooning scores, (C) correlation of vaccenic acid (18:1n7c) with liver inflammation scores, and (D) correlation of vaccenic acid (18:1n7c) with fibrosis scores. Fatty acid levels are presented as a percentage of the phospholipid fraction. Statistical significance was done using ANOVA test and then pairwise comparison using Tukey’s test was performed. Spearman correlation was calculated using GraphPad Prism 6 and Bonferroni correction was used to select significant correlations.</p